In situ fluorometer using a synchronous detector

ABSTRACT

Disclosed herein is a self-contained submersible fluorometer designed for the continuous in situ recording of concentrations of materials in an aqueous environment, said materials being stimulated to fluoresce when excited by light of proper wavelengths.

D United States Patent {151 3,649,833

Leaf [4 Mar. 14 1972 [54] IN SITU FLUOROMETER USING A SYNCHRONOUSDETECTOR R f r Cit d [72] Inventor: William Benjamin Leaf, SilverSpring, Md. UNITED STATES PATENTS [73] Assignees: PrototypesIncorporated, Kensington, Md.; 3,490,875 1/1970 Harmon et a1 ..250/43.5R X Zone Research Incorporated, Washington, DC. part interest to eachPrimary Examiner-Jarnes W. Lawrence Assistant Examiner-Morton J. Frome[221 Attorney-Sughrue, Rothwell, Mion, Zinn & MacPeak 21 A LN 134,781 1pp 0 [57 ABSTRACT 52] US. Cl. ..2s0/71 R, 250/435 R, 250/835 A, Disfflmdis a submelsible flmmmete' 250/833 H 350/96 R 356/246 designed for thecontinuous In situ recording of concentra- [51] Int. Cl 6 21/26 tions ofmaterials in an aqueous environment, said materials Field of Search"250/71 71 43.5 435 being stimulated to fluoresce when excited by lightof proper 250/715, 833 H, 836 FT, 835 A; 350/96 R;

wavelengths.

356/246 4Claims,4DrawingFigures REFERENCE- REF AMPUHER usnr FROM NDLAMPZ DETECTOR WAVESHAPER l R i SYNCHRONOUS AND OPTIC 1 DETECTORINTEGRATOR FIBER l6 WWW 14 m2 3, s49 8 33 SHEET 1 OF 2 INVENTOR WILLIAMB LEAF Zia sf MM QOLK ATTORNEYS BY flu rw KM, kim

PATENTEBPAR I 4 IIM2 3,649 833 SIIEET 2 [IE 2 I A w h W M a 4 WREFERENCE-PT REF AMPLIFIER /30 I LIGHT FROM AND LAMP2 DETECTOR IWAVESHAPER I 28 I 3! 32 34 36 I LIGHT FROM E Z PHOTO I SYNCHRONOUSINTEGRATOR I MOOPTIC DETECTOR OETECTOR FIBER I6 I I AUTOMATIC I GAINCOMT I WOSITIONPOTENTIOMETER K Q REFERENCE AMPLIFIER SHAPER OUTPUT LIGHTFROM FLUORESCENCE PHOTO OF SAMPLE MATERIAL M OETECT R W I I O 28 4 4LIGHT FROM OPTIC P OUT UT FIBER BALANCE PATH-l6 II DETECTOR 34 I I IMPUT\/v V OETECTOR 54 I I OUTPUT INTEGRATOR 3s OUTPUT I I IDRIVESSERVOAMPUFIER) IN SITU FLUOROMETER USING A SYNCI-IRONOUS DETECTOR BACKGROUNDOF THE INVENTION The invention is in the field of fluorometers and morespecifically in the field of fluorometers used to measure concentrationsof fluorescing material, said concentration measurements being used tostudy the effects of pollutants and man's activities in the aqueousenvironment.

The basis of fluorometer measurements to detect material concentrationsis that certain materials exhibit unique, repeatable fluorescentcharacteristics. In particular, when excited by light of certainwavelengths, these materials respond by emitting light in another,longer wavelength band. By selecting proper light sources, opticalfilters and optical detectors, materials which exhibit fluorescence canbe detected and their concentrations measured with a great degree ofaccuracy over a wide range of concentrations in various naturalbackgrounds.

The detectability of the unique fluorescent signatures of variousmaterials in solution forms the basis for the application of thefluorometer to studies directed at the solution to water pollutionproblems. Fluorometer measurements are extremely useful in the area ofwater pollution studies from two aspects. First, by injecting samples ofa fluorescent tracer material into a body of water, studies may be madeof the dispersion and dilution rates of aqueous contaminant fields.Thus, fluorometer measurements can be used to predict the effect of theinjection of contaminant fields into the water body. Second, it ispossible to determine the effects of pollution or human activities onthe ability of a body of water to support a standing crop ofphytoplankton which is a basic link in the marine food chain. This isachieved by the measurement of fluorescent chlorophyll which is found inthe phytoplankton and forms a sensitive indicator of the presence anddensity of phytoplankton in the aqueous environment under study.

In the past, such studies have been attempted by pumping samples of thebody of water under consideration to surface fluorometers or by thecollection of discrete deep samples by submerged sampling bottles.However, there are many disadvantages with these prior methods. First,pumped samples become mixed or smeared in the pumping hose and theresolution and sensitivity of a surface fluorometer to detect smallscale concentration gradients is lost. Further, it is extremelydifficult to make continuous concentration profiles at depths greaterthan about meters. Finally, it has been impossible to assess the changesin sample characteristics occasioned by pumping forces and by pressurechanges and temperature variations encountered by the sample duringtransit to the surface.

The above problems are overcome completely by the in situ fluorometer ofthis invention which provides real-time, continuous measurementcapabilities. The in situ fluorometer operates within the aqueousenvironment itself thus eliminating the requirement of bringing a sampleof water to the surface. The fluorometer disclosed herein permitsconcentration measurements at depths of 200 meters and beyond,permitting dispersion and dilution studies at depths greater than 15meters where bottom effects are critical to understanding localdispersion characteristics.

Further, in the measurement and analysis of concentrations ofphotosynthetic phytoplankton a deep measurement capability isparticularly useful as the depth of the photic zone, in which solarenergy is utilized in the photosynthesis process, frequently exceeds 50meters and the phytoplankton drift or are mixed to considerably deeperdepths.

SUMMARY OF THE INVENTION The instrument of this invention provides aself-contained submersible fluorometer for the continuous in siturecording of concentrations of fluorescing material in a body of waterunder study. The fluorometer employs the double beam optical bridgeprincipal which permits the measurements of very low fluorescent levelswhich occur when measuring relatively small concentrations of materialsamples in large bodies of water.

Utilizing this principal, a light chopper permits a photodetector tolook alternately at the emission from the fluorescing sample and areference light in a standard balancing path of the bridge. Thereference light in the balancing path is adjusted by a servo driven,circular neutral density wedge filter which has a continuoustransmissibility variation of several decades of light. Thephotodetector output is demodulated by a synchronous detector, using asa synchronous reference signal, light derived from the exciting lightsource chopped by the rotation of the light chopper. The detector outputis used to energize a servo motor. The polarity of the motor drivevoltage is such that the circular neutral density wedge filter isrotated in the direction required to maintain a light balance in theoptical bridge. Coupled to the servo motor and the neutral density wedgefilter is a position potentiometer with a wiper arm geared to theneutral density filter. The output voltage from the positionpotentiometer is a potential proportional to the intensity of thefluorescence which is indicative of the concentration of the samplematerial.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the basic externalconfiguration of the fluorometer of this invention with a portion of theouter cover removed to illustrate a section of the internal fluorometerstructure;

FIG. 2 illustrates the internal structure of the in situ fluorometer;

FIG. 3 is a schematic diagram of the electrical components of thefluorometer; and

FIG. 4 is a timing diagram corresponding to the operation of thecircuitry of FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 illustrates thebasic configuration of the in situ fluorometer of the invention. Thefluorometer is generally cylindrical in shape having a faired nose piece9 defining an undulating internal passageway or duct 5 through whichwater is caused to flow. The instrument can either be towed behind amoving boat or placed at rest in a flowing stream with the faired noseportion of the instrument facing the current. In either case, thepressure difference between the intake duct 1 and the outlet duct 3causes water to flow through a transparent cuvette 6. Between the inlet1 and the outlet 3 is positioned a curved duct 5 for carrying the watersample to and from the cuvette 6. The duct 5 is internally blackened andis so shaped to block ambient light from reaching the cuvette.

Filter 4 passes light from an excitation source which emits radiation atwavelengths which excite the sample material, whose concentration is tobe detected, to fluorescence. As the material fluoresces, filter 8passes only those wavelengths of light which correspond to thecharacteristic fluorescence of the sample material.

Coupled to the instrument is cable 7 which may serve as a towing cable,an information carrying line for transferring to the surface thedetected concentration information, and for carrying operating powerfrom a surface power supply to the instrument. It is, of course,possible for the concentration information to be recorded on board thefluorometer by including therein a suitable recorder. Further, thefluorometer can be totally self-contained by including a battery withinthe cylinder 11 for operating the instrument. Utilization of a batterywithin the instrument alleviates the requirement of transferring powerfrom the surface through the cable 7 to the fluorometer. The cable maybe removably attached to cylinder 11 by a water proof connector.

The double beam optical bridge of this invention and its operation willnow be described with reference to FIGS. 2, 3, and 4. As watercontaining the sample material under study passes through transparentcuvette 6, light from lamp 2 passes through excitation filter 4 excitingthe material to fluorescence. Lamp 2 may be a fluorescent lamp or anyother light source which emits radiation containing wavelengths suitablefor exciting the material under study. Excitation filter 4 permits thepassage of only those wavelengths suitable for exciting the material.The resulting fluorescent light as well as some light from filter 4impinge emission filter 8. Filter 8 is selected to pass only thosewavelengths from the desired fluorescent light to the photodetector 28.

The light chopper comprises a light shutter 24l rotated by shutter motor26. The underside of the shutter is reflective to incident light. Thus,when the shutter is in the position shown in FIG. 2, the light outputfrom filter 8 is blocked from the photodetector 28, while the lightoutput from optic fiber 16 is reflected onto the photodetector by theunderside of the shutter. Optic fiber I6 is positioned between the lightshutter 24 and the neutral density wedge filter 18 such that the inputto the optic fiber is controlled by transmissibility of the wedgefilter. Thus, optic fiber 16 provides the balancing path, with the lighttraveling therethrough acting as the reference light for the double beamoptical bridge.

The light for the balancing path is transmitted by optic fiber 14, oneend of which is positioned to receive light from lamp 2 with the otherend being positioned to emit light toward the underside of the wedgefilter opposite the lower end of the optic fiber 16. It should beunderstood that optic fiber 14 can be deleted by utilizing a secondlight source radiating directly on the underside of the wedge filter 18.

When the bridge is balanced, wedge filter 18 is positioned such that theintensity oflight at the output of optic fiber 16 is equal to theintensity of the fluorescent light originating in cuvette 6 and passingthrough filter 8. The position potentiometer 22 is geared to the wedgefilter so that its output potential uniquely defines the wedge filterposition. Since the position of the wedge filter is correlatible to theintensity of the fluorescence, and since the amount of fluorescence isproportional to the concentration of the sample material in the waterflowing through cuvette 6, the output potential of the potentiometer isthus proportional to the sample concentration.

By continuously plotting the output of the potentiometer 22 as thefluorometer is caused to move either vertically or horizontally throughthe body of water under study, vertical and horizontal profiles ofmaterial concentrations can be obtained for use in circulation anddilution studies. For example, to predict the dispersion of aqueouscontaminant fields in a body of water, a tracer dye such as rhodamine Bdye can be added to the aqueous environment. Filter 4 is then selectedto pass radiation which excites the rhodamine to fluorescence whilefilter 8 is selected to pass the wavelength band characteristic offluorescing rhodamine.

Since the dispersion of the tracer dye will generally correspond to thedispersion of a contaminant injected into the water as industrial waste,an accurate prediction of the dispersion of such industrial waste withinthe body of water can be made.

On the other hand, if the concentration of phytoplankton is to bedetermined, one need only replace excitation filter 4 with a filterwhich passes radiation over those wavelengths which excite chlorophyllto fluorescence. Emission filter 8 is selected to pass those wavelengthscharacteristic of fluorescing chlorophyll. Photodetector 28 is selectedto be sensitive to all the wavelengths under consideration. That is, inmaking concentration measurements the photodetector is sensitive to thewavelengths emitted by the excitation lamp 2 as well as the wavelengthswhich pass through the emission filter 8.

The operation of the light bridge will now be described with particularreference to FIGS. 3 and 4. Common numerical destinations in FIGS. 2 and3 identify common elements. Thus, photodetector 28 in FIG. 2 correspondsto the like identified photodetector in FIG. 3.

As shutter 24 rotates under the control of motor 26 the input tophotodetector 28 alternately receives light: from the optic fiber l6 andthe emission filter 8. The output of the photodetector is coupled to acontrol circuit 12. The control circuit is shown in detail in FIG. 3.The photodetector output is coupled to a preamplifier 31. Thus, theoutput from the photodetector is amplified and passed to a bandpassamplifier 32. The output from amplifier 32 appears as the input tosynchronous detector 34. A synchronous reference signal is derived froma reference photodetector 10 which receives light from the excitationlamp 2. The light which impinges reference detector 10 is chopped by thelight shutter 24 as it rotates under the control of the shutter motor26. The synchronous detector output is coupled to an integrator 36 andan automatic gain control circuit 40. The automatic gain control circuitis connected in a feedback loop to the bandpass amplifier 32, and actsto maintain a constant reference or DC level in the output of theamplifier. The output of the integrator is supplied to a diiferentialservo amplifier 38 which controls the rotation of servo motor 20. Servomotor 20 is mechanically coupled to the neutral density wedge filter l8and to the wiper arm of the position potentiometer 22. The potentiometerwiper arm is electrically coupled by line 25 to a recorder 42 whichrecords the potentiometer output potential.

Let it be assumed that the sample material concentration is initially atsome low level as illustrated in FIG. 40. As shutter 24 rotates,chopping the input light to reference photodetector III, a synchronizingsignal is generated and applied to the amplifier and wave shapercircuitry 30. The output of circuit 30 is the square wave illustrated inFIG. 412. With the concentration of the sample material at a constantlevel for a time sufficient to balance the optical bridge, the output ofthe photodetector 28 appears as a generally constant level waveform(FIG. 41."). The output from the photodetector 28 passes through thebandpass filter-amplifier 32 to produce an input to the synchronousdetector 34 as illustrated in FIG. 4a. The output of the detectorappears as illustrated in FIG. 4e. The integrator acts to integrate theoutput from the detector 34 to produce a waveform as illustrated in FIG.4 Synchronous detector circuits are generally known in the art and afull description thereof is unnecessary for an understanding of theinvention. They generally function to produce an output signalindicative of the mismatch or difference between the two portions of amixed input signal that has been demodulated in accordance with asynchronizing signal.

Let it now be assumed that there is a rapid in the concentration of thefluorescing sample material in the body of water through which thefluorometer is passing. This is illustrated in FIG. 4a by a stepincrease in the sample material concentration. Since an increase insample material concentration causes a corresponding increase in theintensity of fluorescence, there is a rapid rise in the output from thephotodetector 28 during those periods when it is viewing the light fromthe cuvette, thus causing a fluctuating signal to appear at the input tothe detector 34 as shown in FIG. 4d.

This results in a pulse train at the output of detector 34, as shown inFIG. 4e, whose magnitude decreases as bridge balance is restored. As aresult, the output from the integrator begins to increase causing animbalance between the inputs to the differential servo amplifier 38. Theservo motor 20 begins to rotate in a direction to drive the circularneutral density wedge filter 18 in a direction required to restore alight balance in the optical bridge. Thus, in this example, the servomotor would rotate in a direction to cause the transmissibility of thewedge filter 18 to increase in the area between the optic fibers l4 andI6. In addition, as the servo motor rotates, the wiper arm of positionpotentiometer 22 moves in the direction to balance the inputs to thedifferential servo amplifier 38. Thus, as seen from FIG. 40 the outputfrom photodetector 28 corresponding to the light from the optic fiber 16increases in response to the detection of an increase in fluoresence ofthe sample material. As the intensity of light in the balancing pathincreases, the intensity difference in the two arms of the bridgedecreases thus explaining the decaying waveform in FIG. 4d. As a result,there is a corresponding decrease in the output from detector 34.

As previously described, the automatic gain control circuit 40 assuresthat the output of the bandpass amplifier 32 assumes a constantreference level when the bridge is balanced. Integrator 36 may be aresistance-capacitor circuit which stores energy received from thedetector. When the output of the detector falls back to its referencelevel in response to the balancing of the optical bridge, the output ofthe integrator levels off at some new value determined by the energystored in the integrator circuitry.

The output potential from the position potentiometer 22 is recorded bycoupling the wiper arm of the potentiometer to a suitable recordingmeans 42 through cable 25. When the recording means is contained withinthe fluorometer, cable 25 runs directly to the recording means. However,if the recording means is located on the surface, cable 25 isincorporated into cable 7.

In an actual embodiment of the complete fluorometer ofthe invention, thecylindrical housing 11 may be provided with diving fins or depressors,as is known in the art, to achieve a desired towing depth and stabilitywhen in use.

While the invention has particularly shown and described with referenceto a preferred embodiment thereof, it will be understood by thoseskilled in the art that various changes in form and detail may be madetherein without departing from the spirit and scope of the invention.

What is claimed is:

1. In an apparatus for measuring the light generated by fluorescingmaterials suspended in a liquid medium and including a dual light pathoptical bridge, a source oflight for illuminating both paths, atransparent sample chamber disposed in one path for carrying the liquidmedium, means for restricting the light entering the sample chamber to aselected wavelength, means for filtering the light leaving the samplechamber to a selected wavelength, servo means disposed in the other pathfor varying the optical transmissivity thereof, a photodetector forsensing the light leaving both paths, a light chopper disposed betweenthe ends of both paths and the photodetector to alternately direct lightfrom both paths to said photodetector, and electrical circuit meansresponsive to the photodetector output for controlling the servo means,the improvement characterized by:

a. the light chopper comprising a motor driven rotating shutter,

b. the electrical circuit means comprising a synchronous detector, and

c. a reference signal photodetector positioned such that received lightis chopped by said rotating shutter for supplying a synchronizing signalfor providing the phase reference to the synchronous detector.

2. The apparatus of claim 1 wherein said servo means includes a servomotor coupled to said electrical circuit means and a neutral densitywedge filter coupled to said servo motor for rotation therewith to varythe illumination in the other light path.

3. The apparatus of claim 2 wherein said servo means further includes aposition potentiometer coupled to said neutral density wedge filter, theoutput of said potentiometer being proportional to the position of saidwedge filter.

4. The apparatus of claim 3 wherein said other light path is defined bya fiber optic light conducting tube.

1. In an apparatus for measuring the light generated by fluorescingmaterials suspended in a liquid medium and including a dual light pathoptical bridge, a source of light for illuminating both paths, atransparent sample chamber disposed in one path for carrying the liquidmedium, means for restricting the light entering the sample chamber to aselected wavelength, means for filtering the light leaving the samplechamber to a selected wavelength, servo means disposed in the other pathfor varying the optical transmissivity thereof, a photodetector forsensing the light leaving both paths, a light chopper disposed betweenthe ends of both paths and the photodetector to alternately direct lightfrom both paths to said photodetector, and electrical circuit meansresponsive to the photodetector output for controlling the servo means,the improvement characterized by: a. the light chopper comprising amotoR driven rotating shutter, b. the electrical circuit meanscomprising a synchronous detector, and c. a reference signalphotodetector positioned such that received light is chopped by saidrotating shutter for supplying a synchronizing signal for providing thephase reference to the synchronous detector.
 2. The apparatus of claim 1wherein said servo means includes a servo motor coupled to saidelectrical circuit means and a neutral density wedge filter coupled tosaid servo motor for rotation therewith to vary the illumination in theother light path.
 3. The apparatus of claim 2 wherein said servo meansfurther includes a position potentiometer coupled to said neutraldensity wedge filter, the output of said potentiometer beingproportional to the position of said wedge filter.
 4. The apparatus ofclaim 3 wherein said other light path is defined by a fiber optic lightconducting tube.